This project will explore the regulatory system that controls progression through the stages of the eukaryotic cell division cycle. Cell-cycle progression is governed by an important class of protein kinases called the cyclin- dependent kinases or Cdks. In the proposed studies, biochemical and molecular genetics approaches will be used in the budding yeast Saccharomyces cerevisiae to address the mechanisms by which Cdks control progression through the final stages of cell division - with an emphasis on the roles of protein substrates that are phosphorylated by Cdks in the cell. In the previous funding period, large numbers of candidate Cdk substrates were identified and characterized, and future work will address the function of selected targets in the control of two major processes: chromosome segregation in mitosis and cell division in cytokinesis. The first aim centers on the Cdk substrate Eco1, which promotes the cohesion of sister chromatids as they are synthesized in S phase. Previous work revealed that Eco1 is inhibited after S phase by Cdk1-mediated phosphorylation, which triggers Eco1 destruction via the ubiquitin ligase SCF-Cdc4. The first aim of the current proposal is to obtain a comprehensive understanding of Eco1 regulation by phosphorylation, both by Cdk1 and by another protein kinase, Cdc7, that was uncovered as an Eco1 regulator in preliminary studies. The second aim of the proposed work will be to understand the regulatory mechanisms that control the initiation of cytokinesis, with a focus on how the dephosphorylation of several Cdk1 substrates helps govern the assembly and function of the contractile ring that drives the division of the cell. The information gained from these studies will provide important new insights into the control of cell-cycle progression, and will thereby enhance our understanding of diseases, such as cancer, in which cell-cycle control or chromosome segregation is defective. PUBLIC HEALTH RELEVANCE: When a cell reproduces, the chromosomes are first duplicated and then distributed into a pair of daughter cells. Errors in this process can result in genetic damage or defects in chromosome number, which can accelerate cancer progression or cause developmental defects. The proposed studies focus on a class of proteins, called cyclin-dependent kinases, that are the master regulators of the steps in cell division. These studies will lead to a better understanding of how errors in chromosome behavior and cell division can arise in human disease.